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Molecular and Cellular Biochemistry

, Volume 404, Issue 1–2, pp 271–279 | Cite as

SEAP activity serves for demonstrating ER stress induction by glucolipotoxicity as well as testing ER stress inhibitory potential of therapeutic agents

  • Raji Lenin
  • Viswanathan Mohan
  • Muthuswamy BalasubramanyamEmail author
Article

Abstract

Endoplasmic reticulum (ER) stress is emerging as a unifying paradigm and one of the underlying mechanisms in the genesis of diabetes and its complications. While this has prompted the development of ER stress inhibitors, there is a limitation in monitoring of ER stress in vitro and in vivo by reliable methodologies. We validated the secreted alkaline phosphatase (SEAP) activity as a surrogate marker of ER stress in mouse β-TC6 cells exposed to glucolipotoxicity or tunicamycin and studied insulin secretion along with alterations in ER stress markers. SEAP activity assay was measured using the Great EscAPe SEAP kit, insulin levels were determined by Mercodia reagents and mRNA expression of ER stress markers was quantified by real-time PCR. SEAP activity in β-cells was significantly decreased (indicating increased ER stress) on exposure either to glucolipotoxicity or tunicamycin. This was accompanied by an increased mRNA expression of ER stress markers (GRP-78, PERK, IRE1α, ATF6, XBP-1, and CHOP) and decreased insulin secretion. Treating the cells with phenylbutyric acid normalized SEAP activity, decreased mRNA expression of ER stress markers and improved insulin secretion. Interestingly, cells exposed to different classes of anti-diabetes agents or compounds such as resveratrol resisted ER stress. Methylglyoxal also induces ER stress and this was counteracted by aminoguanidine. Out study demonstrates SEAP activity as a novel ER stress monitoring assay to investigate the therapeutic value of agents with ER stress inhibitory potential. Future studies should focus on the exercise of adopting this reporter assay for high-throughput screening mode of drug discovery.

Keywords

ER stress SEAP activity UPR Glucolipotoxicity PBA Metformin Vildagliptin β-Cells 

Abbreviations

ATF-6

Activating transcription factor-6

CHOP

CCAAT/enhancer-binding homologous protein

GRP-78

Glucose-regulated protein-78

IRE-1α

Inositol-requiring enzyme-1α

PERK

PKR-like ER kinase

XBP-1

X box binding protein1

PBA

4-Phenylbutyric acid

SEAP

Secreted alkaline phosphatase

PDI

Protein disulfide isomerase

ERO1

ER oxidoreductase1

INSIG1

Insulin-induced gene1

SREBP

Sterol regulatory element-binding transcription factor

AGEs

Advanced Glycation Endproducts

Notes

Acknowledgments

This work was supported by research Grants from the Department of Biotechnology (DBT) and the Department of Science and Technology (DST), Government of India, New Delhi, India. RL acknowledges the financial assistance (Senior Research Fellowship) from the Council of Scientific and Industrial Research (CSIR), New Delhi, India.

Conflict of interest

None.

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Copyright information

© Springer Science+Business Media New York 2015

Authors and Affiliations

  • Raji Lenin
    • 1
  • Viswanathan Mohan
    • 1
  • Muthuswamy Balasubramanyam
    • 1
    Email author
  1. 1.Department of Cell and Molecular BiologyMadras Diabetes Research Foundation & Dr. Mohan’s Diabetes Specialties CentreChennaiIndia

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